Cell movements are critical features of normal embryonic development and tissue homeostasis. Cell migration also drives tumor dissemination and contributes to autoimmune disease. In addition, successful regenerative medicine will require that we know how to control the assembly of cells into functional three-dimensional (3-D) architectures. It is therefore important that we uncover and harness the mechanisms that govern cell movements. Our basic understanding of how cells move derives from studies of cultured cells moving on hard surfaces in vitro. However cells appear and behave differently when cultured in more complex and compliant, 3-D environments. Moreover, in vivo, many cells move as interconnected groups. To elucidate the mechanisms regulating such a collective cell migration in vivo, my laboratory has developed a genetically tractable model: the migration of the border cells in the Drosophila ovary. Over the last >20 years we identified the molecular pathways that control the timing and direction of movement, as well as the critical contributions of key regulators of the actin cytoskeleton, such as the 21kD GTPase Rac. More recently we developed organ culture and live imaging approaches, which enable us to use innovative tools, such as photo-activatable analogs of Rac (PA-Rac) and F?rster Resonance Energy Transfer (FRET). Using these techniques we discovered novel properties of Rac and its relative Cdc42, including unexpected synergistic effects of the two proteins, and the instructive role of Rac in collective cell behaviors. Here we propose to build upon this foundation of genetic screening, live imaging, PA proteins and FRET probes, to test iconoclastic hypotheses concerning the relationships between Rac, Rho, and Cdc42 in migrating cells. In Aim 1, we propose to test the hypothesis that Rac and Cdc42 have redundant effects, in addition to their well-characterized non-redundant and newly discovered synergistic effects. In Aim 2 we will elucidate the molecular mechanisms underlying their synergy. In Aim 3 we propose to investigate the relationship between Rac and Rho. In Aim 4 we propose to elucidate the molecular mechanisms by which cells of a migrating group sense and communicate directional information between the migratory cells, to achieve coordinated collective chemotaxis. Each of these aims is founded upon substantial published and unpublished preliminary data. This work is significant because Rho, Rac and Cdc42 are key nodes in the signaling and cytoskeletal networks that control cell shape and movement. So understanding their mechanisms of action and inter-relationships in vivo is of fundamental importance to cell and developmental biology. Yet there remain key unanswered questions concerning how they function in concert to coordinate collective cell migration. In addition, our studies enhance our ability to understand and control cell movements in therapeutic settings, such as tumor metastasis and tissue engineering. The proposed projects are innovative because they take advantage of a unique combination of cutting edge tools that we have developed, to test new ideas concerning the overlapping and synergistic roles of Rho family proteins in collective cell migration in vivo.